DNA plasmids

ABSTRACT

Disclosed are novel circular DNA plasmids useful as vectors in recombinant methods to secure high levels of E.coli expression of exogenous genes. Plasmids of the invention comprise discrete DNA sequences operative to: (1) confer upon the plasmid the capacity for autonomous replication in a host cell; (2) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained; (3) stabilize maintenance of the plasmid in host cell populations; (4) direct synthesis of a protein product indicative of plasmid maintenance in a host cell population; (5) provide, in series, a plurality of restriction endonuclease recognition sites, unique to the plasmid and facilitative of exogenous gene DNA sequence insertion; and (6) terminate mRNA transcription of adjacent DNA sequences and situated so as to terminate transcription of exogenous gene sequences inserted within the plasmid at said unique restriction endonuclease restriction sites. Plasmids preferably have a size of less than 5.0 kilobases (exclusive of any inserted exogenous gene) and optionally include a DNA sequence operative to provide a strong promoter of mRNA transcription functionally associated with a temperature sensitive repressor sequence. A presently preferred embodiment of novel plasmids of the invention is plasmid pCFM414 (A.T.C.C. No. 40076).

This is a continuation-in-part application of co-owned, co-pending U.S.patent application Ser. No. 521,964, filed Aug. 10, 1983 and nowabandoned.

BACKGROUND

The present invention relates generally to recombinant methods andmaterials for effecting the microbial production of useful polypeptidesand more particularly to novel DNA plasmids useful in securingexceptionally high levels of expression of exogenous genes in E.colihost cells.

Numerous attempts have been made to optimize the extent to whichexogenous genes are subject to expression in microbial host cellpopulations such as E.coli, B.subtilis, yeast and other microbial celltypes. Among the initially more promising developments in theoptimization of E.coli expression of "foreign" genes was isolation ofthe so-called "temperature dependent", "temperature sensitive", or"runaway mutant" forms of circular DNA plasmid. See, generally, Uhlin,et al., Gene, 6, pp. 91-106 (1979); Uhlin, et al., J.Bacteriol., 148,pp. 386-390 (1981); Uhlin, et al., U.K. Pat. No. 1,557,774. Theseautonomously-replicating plasmids could generally be maintained inE.coli host cells in moderate copy numbers when the cells were culturedat a "permissive" temperature below 34° C., e.g., at about 30° C. Uponelevation of culture temperatures to a "non-permissive" level of 37° C.and above, inherent controls on autonomous replication of the plasmidwithin the hose were lost and replication of the plasmid was said to"run away" until quite substantial numbers of copies of the plasmid werepresent in the cell.

The potential utility of such temperature sensitive runaway mutantplasmids in recombinant methods for securing E.coli expression ofexogenous gene products was manifest. Because it is often the case thatexpression of exogenous genes results in formation of products which aretoxic or otherwise detrimental to cell metabolism and because yields ofexogenous gene products are often diminished by host cell degradation ofthe foreign polypeptides over a period of time, it was believed thatoverall yields of desired products could be optimized by delayingreplication of recombinant plasmids until host cells had reached theirmaximum cell culture densities in the fermentor. In this way large scaleDNA transcription and mRNA translation required for host expression of adesired adventitious gene could be temporally regulated to occur at atime close to final product harvest, when host cell detriment andproduct degradation would have minimal effects of overall productyields.

The first of the temperature sensitive runaway mutant plasmids to beisolated, however, suffered numerous drawbacks which made themunattractive as potential expression vectors for use in securing largescale microbial synthesis of commercially significant proteins. First ofall, the plasmids were quite large (averaging around 10 or 12 Kb insize) and their runaway replication in host cells thus constituted asubstantial energy drain. This difficulty was alleviated in part uponthe development of smaller sized, "mini-plasmids" such as the 4.6 KbpKN402 (Deutsche Sammlung von Microorganismen, "DSM" Accession No. 1228)but was exacerbated even for the mini plasmids upon insertion of one ormore "marker" genes coding for a protein product indicative of plasmidmaintenance in the host (e.g., a gene for coding for β-lactamaseproduction resulting in transformed host cell phenotypic resistance toampicillin). Plasmid pKN402-derived plasmids including marker genesinclude, e.g., the ampicillin-resistance conferring plasmid pKN403 (DSMAccession No. 1229), plasmid pKN404 conferring streptomycin resistance,and plasmids pMOB45 and pMOB48 [10.5 Kb and 9.5 Kb, respectively, seeBittner, et al., Gene, 15, pp. 319-329 (1981)] which conferchloramphenicol and/or tetracycline resistance. With the insertion ofeven a moderately-sized exogenous gene with a functionally associatedpromoter/regulator sequence the plasmid size could easily extend to 11or 12 Kb. Large plasmid size and attendant host cellular energy drainsupon runaway replication result in significant problems in view of therelatively high "basal" copy numbers and the enormously high "runaway"copy numbers for a pKN402 and many of its derivatives. U.K. Pat. No.1,557,774, for example, reports 50 plasmid copies per cell (a "copynumber" of 25) for pKN402-tranformed E.coli at 30° C. and amplificationup to about 5000 plasmid copies per cell upon shift in cell culturetemperature to 40°. While such high copy numbers are advantageous in thesense of favoring extensive mRNA transcription of exogenous genes, muchof the total cellular energy is wasted in DNA replication and mRNAtranscription of DNA sequences having little, if any, consequence to thegoal of exogenous gene expression. Depletion of cellular energyresources, of course, has a direct and unfavorable influence on the rateof mRNA translation into desired protein products.

The problems associated with relatively high basal copy numbers ofcommon temperature sensitive runaway mutant plasmids take on evengreater significance upon consideration of potential exogenous geneproduct toxicity because even at low ("permissive") temperatures thehigh rates of transcription and translation events can providequantities of gene product which interfere with optimal host cell growthin culture. Manipulations geared toward reducing the basal and amplified(elevated temperature induced) copy number of runaway mutant plasmidshave generally resulted in either loss of temperature sensitivitycharacteristics or loss of the capacity for maintenance of the plasmidsin host cells. See, e.g., Hashimoto-Gotoh, et al., Gene, 16, pp. 227-235(1981). Of interest to the background of the present invention is theindependent notation of the existence of "partition" DNA sequences inpSC101-derived plasmids, which sequences reportedly facilitate stableplasmid inheritance. See, Meacock, et al., Cell, 20, pp. 529-542 (1980).

A partial attempt to deal with the problem of toxic gene product"leakage" occasioned by high basal copy numbers has taken the form ofincorporation of structural gene transcription promoter DNA sequencesfunctionally associated with "strong" repressor (operator) sequenceswhich allow for highly selective chemical or, preferably, thermalcontrol of exogenous gene expression. See, e.g., Sninsky, et al., U.S.Pat. No. 4,374,927; Sninsky, et al., Gene, 16, pp. 275-286 (1981);Remaut, et al., Gene, 22, pp. 103-113 (1983).

The use of "strong" promoter sequences in pKN402-derived plasmids, ofcourse, occasions corresponding potential losses in overall efficiencyof gene expression owing to the lack of correspondingly strong mRNAtranscription termination sequences. In the absence of such sequences,mRNA transcription of exogenous genes will ordinarily be accompanied bya "read through" into adjacent DNA sequences with corresponding cellularenergy drains, possible significant interference with exogenous genemRNA binding to ribosomes (owing to "oversizing") and, in the case ofthe temperature sensitive runaway mutants, possible interference withproper transcription of DNA sequences essential to autonomousreplication of the plasmid and the runaway characteristic.

Another disadvantage of pKN402-derived plasmids has been the generallack of unique restriction endonuclease enzyme recognition sites whichwould allow ready incorporation of exogenous gene sequences. Wherepresent, such unique sites are frequently at positions intermediate amarker gene, effectively necessitating plasmid constructions involvingtwo selectable phenotypic marker genes to allow for both exogenous geneinsertion and verification of host transformation. See, Hashimoto-Gotoh,et al., supra, but cf, Remaut, et al., supra, discussing insertion of a"multilinker sequence" in a 7 Kb pKN402-derived plasmid designated pCP3.

Despite substantial efforts at modification of the originally isolatedrunaway mutants, there continues to exist a need in the art forsmall-sized, autonomously-replicating, stably-maintained, selectable,circular DNA plasmids having temperature sensitive copy number mutantreplication characteristics. Optimal plasmids of this description wouldhave relatively low basal copy numbers (in the range of 1 to 20 andpreferably 1 to 10) as well as relatively low amplified, temperatureelevation-induced, copy numbers (in the range of 100 to an unlimitednumber and preferably 100 to 300) and would thereby avoid both excessivecellular energy drains during maintenance and runaway conditioninduction and premature "leakage" of exogenous gene products intotransformed host cytoplasm. Optimal plasmids would include DNA sequencesoperative to provide for ready incorporation of exogenous genes withoutinterfering with the function of selectable marker genes and would alsoprovide DNA sequences operative as mRNA transcription terminators. Thelatter DNA sequences would be provided at a locus wherein they couldfunction to terminate transcription of inserted exogenous genes,especially those under the control of strong promoter DNA sequenceswhich might optionally be provided in such optimal plasmids.

BRIEF SUMMARY

The present invention provides novel circular DNA plasmids exceptionallyuseful as vectors in recombinant methods for securing high levels ofexpression of exogenous genes.

Plasmids of the invention generally comprise discrete DNA sequencesoperative to: (1) confer upon the plasmid the capacity for autonomousreplication in a host cell; (2) control autonomous plasmid replicationin relation to the temperature at which host cell cultures aremaintained; (3) stabilize maintenance of the plasmid in host cellpopulations; (4) direct synthesis of a protein product indicative ofplasmid maintenance in a host cell population; (5) provide, in sequence,a plurality of restriction endonuclease recognition sites, unique to theplasmid and facilitative of exogenous gene DNA sequence insertion; and(6) terminate mRNA transcription of exogenous gene sequences insertedwithin the plasmid at said unique restriction endonuclease restrictionsites.

Plasmids of the invention preferably have a size of less than 5.0kilobases (exclusive of any inserted exogenous gene), have relativelylow basal copy numbers and optionally include a DNA sequence operativeto provide a strong promoter of mRNA transcription which is functionallyassociated with a temperature sensitive respressor sequence. Presentlypreferred embodiments of novel plasmids of the invention include plasmidpCFM414, which was deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md., on July 21, 1983 in accordancewith the U.S. Patent and Trademark Office's requirements formicroorganism deposits, and designated A.T.C.C. No. 40076; plasmidspCFM424, pCFM510, pCFM511, pCFM512, pCFM516, pCFM517, pCFM526, pCFM536,pCFM636, pCFM736 and pCFM836, whose construction are disclosed in detailherein for duplication by those skilled in the art. In transformedE.coli cells, a basal copy number of about 20 or less is maintained forthese plasmids at temperatures below 34° C.

Plasmids of the invention provide for high level E.coli expression ofexogenous structural genes as exemplified by genes coding for suchdiverse polypeptides as human insulin-like growth factor, humanurogastrone and the alpha subunit of bovine glycoprotein hormone.Numerous other aspects and advantages of the present invention willbecome apparent to those skilled in the art upon consideration of thefollowing detailed description thereof.

DETAILED DESCRIPTION

The present invention provides a class of circular DNA plasmid vectorswhich possess the substantive advantages of temperature sensitive copynumber mutant plasmids currently available in the art but which aresubstantially free of many of the functional defects of such plasmids.Plasmids of the invention further possess characteristics not presentlyavailable in temperature-sensitive runaway plasmids which render themexceptionally suitable for use in securing E.coli expression ofexogenous gene products. Plasmids constructed according to the inventiongenerally comprise a novel combination of physically discrete(non-overlapping), functionally cooperative DNA sequences.

1. Each plasmid of the invention incorporates a DNA sequence operativeto confer upon the plasmid the capacity for autonomous replication inhost E. coli cells. Specifically suitable for use in achieving thisfunction are DNA sequences comprising (1) the gene commonly referred toas "Rep A" and (2) an "origin of replication" ("ori") sequence. The RepA gene codes for E.coli synthesis of an approximately 33,000 daltonprotein whose concentration in E.coli is thought to directly determinethe number of plasmid replication initiation events. See, e.g., Kollek,et al., Mol.Gen.Genet., 162, pp. 51-57 (1978) and Light, et al.,E.M.B.O. Journal, 2, pp. 93-98 (1983). The "ori" sequence includes asequence coding for "Rep A4", a 14,000 dalton protein.

2. Each plasmid of the invention incorporates a DNA sequence operativeto confer thermal sensitive control of plasmid copy number. By way ofexample, a suitable copy number control sequence would include (1) agene coding for "cop B", and (2) a "cop A" sequence The cop B gene codesfor E.coli production of an approximately 11,000 dalton protein whichoperates as a transacting repressor. Cop B is made constitutively in thecell depending on cell growth conditions and functions as a repressor bybinding to a region at or near the promoter for transcription of Rep A.Cop A is a 90 nucleotide RNA-synthesis-directing sequence. TranscribedRNA is made in the opposite orientation to transcription of Rep Aprotein and the cop A overlaps the leader mRNA directing sequence whichprecedes the Rep A sequence. Cop A RNA apparently operates by way of anRNA-RNA interaction that decreases translation of the Rep A protein.See, e.g., Stougaard, et al., Proc.Nat'l.Acad.Sci. U.S.A., 78, pp.6008-6012 (1981); Molin, et al., Mol.Gen. Genet., 181 pp. 123-130(1981); Light, et al., J.Bacteriol., 151, pp. 1129-1135 (1982); Light,et al., E.M.B.O. Journal, 2, pp. 93-98 (1983).

3. Each plasmid of the invention incorporates a DNA sequenceparticipative in maintenance of the plasmid in host cells. Typicallyderived from a stable low copy number E.coli plasmid and referred to asthe par locus within the plasmid, such sequences operate to ensure thatduring host cell division at least one copy of each plasmid present inthe parent cells will be distributed to daughter cells. See, e.g.,Meacock, et al., Cell, 20, pp. 529-542 (1980).

4. Each plasmid of the invention incorporates a DNA sequence operativeto generate a protein "marker" substance which indicates plasmidmaintenance in host cells. Typically, such a sequence codes for aprotein conferring antibiotic resistance.

5. Each plasmid of the invention incorporates a DNA sequence providingat least two and preferably three or more recognition sites forrestriction endonuclease cleavage which are unique to the plasmid (i.e.,not present in any other region of the plasmid) and hence permit readyinsertion of an exogenous gene.

6. Each plasmid of the invention incorporates a DNA sequence operativeas a terminator of mRNA transcription of adjacent DNA sequences andsituated within the plasmid so as to terminate transcription of anexogenous gene inserted within the plasmid. While typically derived from3' untranslated regions of viral genomic DNA, such sequences mightsimply comprise a series of multiple translation stop codons whichappear to function as mRNA transcription terminators in some instances.See, e.g., Gentz, et al., Proc.Nat'l.Acad.Sci.U.S.A., 78, pp. 4936-4940(1981) and Yanofsky, Nature, 289, pp. 751-758 (1981).

All the above discrete functional DNA sequences of plasmids of theinvention are preferably provided with a minumum of interveningnon-functional DNA sequences so that the size of the plasmid may be 5 Kbor less, allowing for optimal efficiency in the management of hostcellular energy resources. Preferred plasmids of the invention may alsoinclude a DNA sequence operative as a promoter of mRNA transcriptionfunctionally associated with a plasmid-borne or host chromosomal DNAsequence operative to control the promoter. Promoter function could bethermally dependent in the same manner as plasmid copy control, e.g., athermal stimulus which would serve to diminish repression of plasmidreplication would serve to derepress the promoter. Optimally, enhancedplasmid replications would occur at a first elevated temperature andderepression of the promoter would occur at a second, higher, elevatedtemperature. Such a promoter/regulator DNA sequence could be situatedwithin plasmids of the invention at a locus adjacent the transcriptiontermination sequence and spaced apart therefrom by the above-notedgene-insertion-facilitating series of unique restriction sitesAlternatively, the regulator sequence could be located on a separateplasmid or carried on host cell chromosomes.

The following examples serve to illustrate construction and use ofplasmids of the invention. More particularly, Example 1 relates toconstruction and characteristics of plasmid pCFM414 according to theinvention; Examples 2 through 4 respectively relate to use of pCFM414 toachieve high levels of E.coli expression of genes for human insulin-likegrowth factors human urogastrone and a bovine glycoprotein hormonesubunit; Examples 5 through 7 relate to construction and characteristicsof alternative temperature sensitive plasmids according to theinvention.

EXAMPLE 1

Plasmid pCFM414 consists of approximately 4465 base pairs and includes asingle EcoRI restriction site, the 3' terminal base pair (C/G) of whichis designated base pair number 1 of the plasmid. (Unless otherwiseindicated, base pair numbers used in conjunction with restriction siteswill indicate the 3'-most initial pair of residues remaining uponcleavage with the designated enzyme.) Set out in Table I below is thesequence of base pairs 1 through 36 of pCFM414.

                                      TABLE I                                     __________________________________________________________________________    EcoRIHpaIXbaINcoIHindIIIXhoIBamHI                                             __________________________________________________________________________     ##STR1##                                                                     __________________________________________________________________________

The Table I sequence is seen to comprise a sequence of base pairsproviding a series of recognition sites for cleavage by restrictionendonuclease enzymes HpaI, XbaI, NcoI, HindIII, XhoI, and BamHI, all ofwhich are unique to pCFM414. (While the manufactured Table I sequencewas designed to include an XbaI recognition site, attempts to cutpCFM414 with the enzyme have not been uniformly successful.

Commencing with bp 37 and extending through bp 235 is a transcriptionterminator sequence. More specifically, the sequence comprises the"Toop" terminator of lambda bacteriophage obtained from plasmid pKO1-T[See, McKenney, et al., Gene Amplif.Anal., 2, pp. 383-415 (1981)] on aMboI fragment to which were added "linker" bases providing a 199 bpfragment with BamHI sticky ends. In the process of incorporation intopCFM414, the "rightward" BamHI site of the Toop-containing fragment wasnot reconstituted and thus the uniqueness of the "leftward" BamHI sitewithin the final plasmid construction was preserved.

The DNA sequence spanning bp 236 through 415 of pCFM414 is a residue ofintermediate construction procedures employed in developing the finalplasmid. The DNA had its origins in plasmid pSM2 [See, Mickel et al.,J.Bacteriol., 127, pp. 644.655 (1976)] as a BC1I to Bg1II fragment. Inthe course of pCFM414 construction, the Bc1I site was lost upon ligationto the BamHI ended fragment (providing bp 37-35) as described in thepreceding paragraph. The entire transcription terminator sequence (bp37-235 and bp 236-415 may be excised from pCFM414 by BamHI and Bg1IIdigestion.

The DNA sequence of pCFM414 spanning bp 416 through 1259 was derivedfrom pKN402 as a partial Bg1II and Sa1I digestion product. The sequenceincludes an E.coli cop B gene and its promoter, the cop A sequence and aportion of the initial sequence of the Rep A gene. The sequence may beremoved from pCFM414 by a partial Bq1II and Sa1I digestion.

The pCFM414 sequence spanning bp 1260 through 2879 was derived aspartial PstI and Sa1I digestion fragment of plasmid NR1 (R100) [See,Rownd, et al., Ann.N.Y.Acad.Sci., 182, pp. 188-206 (1971)]. The PstIsite was blunted off and a synthetic SstI linker was added. The sequenceincludes codons for synthesis of Rep A and also includes an "ori"sequence incorporating the Rep A4 gene. The sequence including bp1260-2879 may be excised from pCFM414 by Sa1I and SstI digestion.

Spanning bp 2880 through 4088 of pCFM414 is a gene coding for E.coliproduction of β-lactamase enzyme which confers an ampicillin resistantphenotype. The sequence was originally derived as an EcoRI and partialHgaI digestion fragment of pBR322. Subsequently an internal sequence ofthis gene (spanning XmnI and Bg1I restriction sites) was replaced by aXmnI to BglI digestion fragment from the β-lactamase gene of plasmidpUC9 [Vieira, et al., Gene, 19, pp. 259-268 (1982)] which does notprovide either a HincII or a PstI restriction site.

Base pairs 4089 through 4465 of pCFM414 comprise a partition regulatingsequence derived from pSC101 (A.T.C.C. 37032) as a HincII and AvaIdigestion fragment. Digestion of pCFM414 eith EcoRI and SstI will yielda single fragment comprising base pairs 4089 through 4465.

Table II, below, provides in tabular form a summary of pCFM414constituents. Restriction endonucluease recognition site designationsappearing in brackets are indicative of those sites associated with thederivation of particular fragments which were destroyed by blunt-endingprior to plasmid construction or "lost" in the course of joining with asticky end that did not restore the complete restriction site.

                  TABLE II                                                        ______________________________________                                        Sequence        Principal Function                                            ______________________________________                                        Base Pairs 0-36 Restriction sites for gene                                    EcoRI-BamHI     insertion                                                     Base Pairs 37-235                                                                             mRNA transcription                                            [MboI]-[MboI]   termination                                                   BamHI-[BamHI]                                                                 Base Pairs 236-415                                                                            "Spacer" and possible mRNA                                    [BclI]-BglII    transcription termination                                     Base Pairs 416-1259                                                                           Cop A and Cop B gene                                          BglII-SalI                                                                    Base Pairs 1260-2879                                                                          Rep A gene and "ori"                                          SalI-SstI       including Rep A4 gene                                         Base Pairs 2880-4088                                                                          β-lactamase gene                                         [HgaI]-[EcoRI]                                                                SstI-[EcoRI]                                                                  Base Pairs 4089-4465                                                                          Stability sequence                                            AvaI-[HincII]                                                                 AvaI-EcoRI                                                                    ______________________________________                                    

While the incorporation of the above-noted specific sequences allowedfor construction of pCFM414 in a size of less than 4.5 kilobases, itwill be apparent that alternative DNA sequences providing identicalfunctional characteristics could be incorporated into plasmids of theinvention although use of available alternative sequences appearsunlikely to be as DNA "conservative".

Transcription termination sequences other than "Toop" derived fromlambda bacteriophage may be suitably selected from among thosedescribed, for example, in Adhya, et al., Ann.Rev Biochem., 47, pp.967-996 (1978). For descriptions of sources of DNA sequences providingthe stabilizing or partition maintenance function other than the "par"locus of pSC101, see, Som, et al., Plasmid, 5, pp. 150 et seq. (1981),Nordstrom, et al., Plasmid, 4, pp. 215 et seq. (1980) and McKell, etal., (Abstract) page 347, J. Supramolecular Structure Supp. No. 4, 9thAnnual ICN-UCLA Symposium (1980). Alternative sources of "ori" and RepA4gene sequences operative to confer autonomous replication of plasmids ofthe invention include those described in Rosen, et al., Mol.Gen.Genet.,179, pp. 527-537 (1981). Plasmid NRl can provide an alternative sourceof a CopA DNA sequence as well as a CopB gene. See, Stougaard, et al.,Mol.Gen.Genet., 181, pp. 116-122 (1981).

Upon transformation of E.coli cells (e.g., K-12 strains AM7, JM103 orthe like), the copy number of pCFM414 within cells maintained at below34° C. (i.e., at 30° C.) is 20. Elevation of culture temperature toabove 34° C. (i.e., to 37° C.) results in uncontrolled replication untilcell death occurs. On the whole, elevation of culture temperature from30° C. to 37° C. will give rise to a doubling in copy number about everyfifteen minutes. Elevation from 30° to 42° C. results in copy numberdoubling about every twelve minutes.

The following example relates to use of pCFM414 to secure E.coliexpression of a gene coding for the alpha subunit of bovine glycoproteinhormone.

EXAMPLE 2

Two plasmids were constructed for use in securing direct E.coliexpression of the alpha subunit common to bovine glycoprotein hormones,and were utilized to examine the effect of pCFM414 on expression of anexemplary gene. A first plasmid, pBα-E₂, contained a cDNA-derivedstructural gene for the alpha subunit polypeptide in association with atrp promoter A second plasmid, pBα-E₃, was constructed by incorporationof the entire promoter and structural gene sequence from pBα-E₂ intoplasmid pCFM414 which had been digested with EcoRI and BamHI.

Growth of E.coli AM7 cells transformed with pBα-E₂ resulted inproduction of approximately 2.5 mg/OD-liter of the desired polypeptide.E.coli AM7 cells transformed with pBα-E₃ were initially grown at 28° C.and the culture temperature was then raised to 37° C. Production of thedesired polypeptide by the pBα-E₃ transformed AM7 cells was estimated bygel chromatography to be approximately 25 mg/OD-liter, or approximatelyten-fold the production of pBα-E₂ transformed AM7 cells.

The following example relates to use of pCFM414 to secure E. coliexpression of a gene coding for the alpha subunit of human urogastrone.

EXAMPLE 3

This example summarizes experimental data set out in co-owned,co-pending U.S. application Ser. No. 486,091, [PCT InternationalPublication No. WO83/04030, published Nov. 24, 1983] entitled "TheManufacture and Expression of Genes for Urogastrone and PolypeptideAnalogs Thereof", filed Apr. 25, 1983 by Banks, et al and now abandoned.Briefly put, two significant plasmids were constructed for use insecuring direct E.coli expression of human urogastrone. A first plasmid,pADH-25, contained a manufactured structural gene for urogastrone inassociation with a trp promoter. A second plasmid, pADH-59, wasconstructed by incorporation of the entire promoter and structural genesequence from pADH-25 into pCFM414 which had been digested with EcoRIand XhoI.

Growth of E.coli JM103 cells transformed with pADH-25 resulted inproduction of approximately 15 micro- grams/OD-liter of the desiredpolypeptide by radioreceptor assay. E.coli JM103 cells transformed withpADH-59 were initially grown at about 30° C. and the culture temperaturewas then raised to 37° C. Production of the desired polypeptide by thepADH-59 transformed JM103 cells was estimated by gel chromatography tobe approximately 50 mg/OD-liter, or well over a hundred-fold increaseover the production by pADH-25 transformed JM103 cells.

The following example relates to use of pCFM414 to secure E.coliexpression of a gene coding for the alpha subunit of human insulin-likegrowth factor.

EXAMPLE 4

This example summarizes experimental data set out in co-owned,co-pending, concurrently-filed U.S. application Ser. No. 521,966,entitled "Microbial Expression of Insulin-Like Growth Factor", filedAug. 10, 1983, now abandoned and its continuation-in-part U.S. Ser. No.633,451, filed July 26, 1984, by Banks, et al now abandoned. Brieflyput, two significant plasmids were constructed for use in securingdirect E. coli expression of insulin-like growth factor. A firstplasmid, pT5-4-IGF-I, contained a manufactured structural gene for thepolypeptide in association with a synthetic promoter. A second plasmid,pADP-223, was constructed by incorporation of the entire promoter andstructural gene sequence of pT5-4-IGF-I into pCFM414 which had beendigested with HindIII and XhoI.

Growth of E.coli JM103 cells transformed with pT5-4-IGF-I resulted inproduction of approximately 2 micrograms per OD-liter of the desiredpolypeptide. E.coli JM103 cells transformed with pADP-223 were initiallygrown at 28° C. and the culture temperature was then raised to 37° C.Production of the desired polypeptide by the pADP-223 transformed JM103cells was estimated by gel chromatography to be approximately 5micrograms per OD-liter, or approximately two-fold the production ofpT5-4-IGF-I transformed JM103 cells.

The following examples relate to alternative construction for a plasmidaccording to the present invention.

EXAMPLE 5

A first series of plasmids was constructed based on pCFM414 andprincipally involving alterations in the copy control regions. Thesewere designated pCFM510, pCFM511, pCFM512, pCFM516 and pCFM517 and theirpreparation is set out below.

A. pCFM510

Plasmid pCFM414 was digested with Bg1II and PstI to delete the(pKN402-derived) Cop A and Cop B regions and the large fragment wasretained. Plasmid NR1 was digested with Bg1II and PstI and the smallfragment containing the NR1 Cop A and Cop B sequences was isolated andligated into the large fragment of pCFM414 to provide pCFM510. Theplasmid therefore principally comprises the entire Cop A, Cop B, "Rep A"and "ori" sequences of NR1 and has six more base pairs (4471) thanpCFM414 (4465).

B. pCFM511

Plasmid pCFM414 was digested with Bg1II and PstI to delete the(pKN402-derived) Cop A and Cop B regions and the large fragment wasretained. Plasmid pSM1, a derivative of pR12 [see, Stougaard, et al.,Molec.Gen.Genet., 181, pp. 116-122 (1981) and Rosen, et al., Molec.Gen.Genet., 179, pp. 527-537 (1980)] was digested with Bg1II and PstI andthe small fragment containing the pSM1 Cop A and Cop B sequences wasisolated and ligated into the large fragment of pCFM414 to providepCFM511. The plasmid therefore principally comprises the Cop A and Cop Bsequences of pSM1 and the "Rep A" and "ori" sequences of NR1 and has sixmore base pairs (4471) than pCFM414 (4465).

C. pCFM512

Plasmid pCFM414 was partially digested with XmnI and PstI to delete the(pKN402-derived) Cop A sequence of pCFM414 and the large fragment wasretained. Plasmid NR1 was digested with XmnI and PstI and the smallfragment containing the Cop A sequence was isolated and ligated into thelarge fragment of pCFM414 to provide pCFM512. The plasmid thereforeprincipally consisted of the Cop A region of NR1, the Cop B region ofBeu 1, and the "Rep A" and "ori" sequences of NR1. The plasmid has thesame number of base pairs (4465) as pCFM414. The promoter thattranscribes the Cop B mRNA has a single base pair mutation (at 435)which is T/A rather than C/G. This mutation is responsible for thetemperature sensitive copy number phenotype.

D. pCFM516 and pCFM517

Plasmid pCFM512 are prepared in part (C) above was subjected tohydroxylamine mutagenesis in order to alter a single base pair (598) inthe Cop B protein coding region from C/G to T/A. Plasmid pCFM516contained this single base pair mutation (effectively changing the aminoacid coded for from alanine to valine). Plasmid pCFM517 contained atleast one as-yet undetermined base pair mutation in the region between848 and 1282.

EXAMPLE 6

Plasmids pCFM414 of Example 1 and plasmids pCFM510, pCFM511, pCFM512,pCFM516 and pCFM517 of Example 5 were screened for plasmid copy numbercharacteristics in exponential and stationary phases of host cell growthas well as for protein product expression.

A protein αCon-1-IFN coding for production of a consensus leucocyteinterferon polypeptide was employed as the "model" sequence forinsertion into the plasmids. The αCon-1-IFN sequence with its precedingShine-Delgarno sequence optimized for translation initiation and with alambda bacteriophage promoter was inserted into each EcoRI/BamHIdigested plasmid.

Each plasmid was co-transformed into an E.coli FM3 host with anotherpSC101-derived plasmid into which had been cloned a 1.1 Kb (Bg1II toPstI) lambda bacteriophage DNA fragment including the gene coding forthe PL repressor protein, CI857. At 42° C., the CI857 protein isinactivated, inducing the PL promoter to initiate transcription.

From fresh overnight cultures in L Broth+50 ug/ml Ampicillin eachplasmid-host system was inoculated into L Broth and grown at 28° C. tomid exponential optical density. (≦1.0 OD_(600nm) BeckmanSpectrophotometer). Cell aliquots (2.0 ODml) were taken for copy numberand protein gel analysis. The cultures were diluted to low celldensities in L Broth and allowed to grow at 42° C. for up to 4 hours.Cell aliquots (2 ODml) were again taken for copy number and protein gelanalysis.

Table III below provides a summary of relative plasmid copy number/hostgenome equivalent at different growth temperatures. Plasmid copy numberis roughly calculated by isolating (alkaline lysate procedure) plasmidDNA from 2 ODml of cell pellet and comparing the amount of DNA/ODml to astandard plasmid with known copy number (pCFM511, copy No.=20).

                  TABLE III                                                       ______________________________________                                        Relative Plasmid Copy Number                                                  Stationary    Exponential Phase                                                       Phase                            at                                   Plasmid at 28° C.                                                                        at 28° C.                                                                       at 37° C.                                                                     at 42° C.                                                                     42° C.*                       ______________________________________                                        pCFM414 200       20       runaway                                                                              runaway                                                                              50                                   pCFM510  4         2        2      2      2                                   pCFM511 40        20       20     20     10                                   pCFM512  4         2       10-20  50-100 --                                   pCFM516 20        10       100-200                                                                              runaway                                                                              100                                  pCFM517 40        20       200-400                                                                              runaway                                                                              100                                  ______________________________________                                         *With P.sub.L --αCon1-IFN gene insert                              

The right-hand column of Table III illustrates the variation in plasmidcopy number obtained when host cells were transformed with the plasmidscontaining the αCon-1-IFN sequence. Analysis of the protein products ofthe host cells revealed that the desired polypeptide constituted 14-17%of the total cell protein of the pCFM510 transformed cells; 25-30% ofthe pCFM511 transformed cells; and 60-70% of the pCFM414, pCFM516, andpCFM517 transformed cells.

EXAMPLE 7

Certain additional plasmids of the invention were designed for thepurposes of (a) increasing the number of unique restriction sitespresent in pCFM414; and/or (b) incorporating a promoter sequence intothe vector with and without a ribosomal loading sequence; and/or (c)changing the ampicillin resistance, β-lactamase marker gene in pCFM414to another antibiotic resistance marker gene. The newly designedplasmids are designated pCFM424, pCFM526, pCFM536, pCFM636, pCFM736 andpCFM836, the preparation of which is as follows:

A. pCFM424

A derivative of plasmid pCFM414, designated pCFM424, consists ofapproximately 4628 base pairs and includes the following:

(1) the entirety of the pCFM414 sequence extending from the base pairs37 through 4465;

(2) a lambda bacteriophage P_(L) promoter; and

(3) an expanded "bank" of restriction endonuclease enzyme recognitionsites as well as an adjacent sequence of base pairs providing multiple"stop" condons.

More specifically, the sequence of 199 base pairs set out in Table IVbelow replaces the sequence of base pairs occupying positions 1-36 ofpCFM414.

                                      TABLE IV                                    __________________________________________________________________________     ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                     __________________________________________________________________________

The Table IV sequence is seen to comprise a sequence of base pairsproviding a series of recognition sites for cleavage by restrictionendonuclease enzymes HgiA, ClaI, HpaI, XbaI, NcoI, HindIII, XhoI, BamHI,and SstII, all of which are unique to pCFM424.

Furthermore, a lambda P_(L) promoter sequence derived from plasmid pKC30[See, e.g., Shimatake, et al., Nature, 292, pp. 128-132 (1981)] as a 130base pair fragment by digestion with Bg1II and HgiA is present inpCFM424 at a locus following the stability gene sequence and precedingthe restriction site "bank". The incorporation procedure involvesaddition of an EcoRI linker to the Bg1II sticky end of pCFM414.

Control over the P_(L) promoter within a pCFM424transformed host may beprovided in a number of ways through use of the C_(I857) repressor gene.As one example, E.coli strain K12ΔHtrp has this gene integrated into thebacterial chromosome and could provide the host. See, Bernard, et al.,Gene, 5, pp. 59-76 (1979). Alternatively, the gene could be isolatedfrom lambda bacteriophage DNA as a 1.1 Kb PstI to Bg1II fragment. Thiscould be inserted into a suitable low copy plasmid which could be"co-transformed" into a suitable host with a pCFM424. The fragment couldalso be associated within a translocation sequence and integrated intothe chromosome of any selected host E.coli strain.

It is expected that plasmid pCFM424 will permit thermally-inducibleexpression of selected exogenous genes and that its use will providequantities of desired polypeptide products in excess even of thoseprovided by pCFM414.

B. pCFM526

Plasmid pCFM 526 is prepared as a derivative pCFM516 and differs from itin terms of deletion of the restriction site bank sequence spanning basepairs 1 through 36 and insertion of a sequence of 223 base pairs set outin Table V.

                                      TABLE V                                     __________________________________________________________________________     ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                     __________________________________________________________________________

The design for pCFM526 is thus seen to include the P_(L) promoter, anexpanded bank of unique restriction sites (vis-a-vis pCFM414) and aribosomal loader sequence intermediate the promoter and the majorcomponents of the restriction bank.

C. pCFM536

Plasmid pCFM536 is also prepared as a derivative of pCFM516 and differsfrom it in terms of deletion of the restriction site bank sequencespanning base pairs 1-36 and insertion of a sequence of 191 base pairsset out in Table VI.

                                      TABLE VI                                    __________________________________________________________________________     ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                    __________________________________________________________________________

The design for pCFM 536 is thus seen to be similar to that of pCFM526except for the absence of the ribosomal loader sequence.

D. pCFM636

Plasmid pCFM636 is prepared as a derivative of pCFM536, constructed toincorporate a Kanamycin resistance marker gene in place of theampicillin resistance marker. The β-lactamase gene is first deleted bydigestion of pCFM536 with SstI and EcoRI (partial, at EcoRI sitepreceding the P_(L) promoter) and the EcoRI site is converted to anAatII site by a linker which "kills" the EcoRI site by omission of theinitial G/C base pair. This serves to delete not only the marker genebut also the entire "par" or stability sequence The Kanamycin genesequence may be obtained as a SmaI to HindIII fragment from the Tn5plasmid of Beck, et al., Gene, 19, pp. 327-336 (1982) or Auerswald, etal., Cold Spring Harbor Symp. Quant. Biol., 45, pp. 107-113 (1981) andprepared for insertion into the large fragment of the above-notedSstI/EcoRI digestion of pCFM536 by addition of a SstI linker to the SmaIsticky end an NdeI linker to the HindIII sticky end. The "par" locussequence may be obtained, as in Example I, as a HincII to AvaI digestionfragment of pSC101. For combination with the Kanamycin gene andinsertion into the large fragment of pCFM536, the HincII end is firsttreated with a Sa1I linker and then an AatII linker. The AvaI site istreated with a BamHI linker and then an NdeI linker. The large fragmentof pCFM536 (AatII/SstI), the Kanamycin gene fragment (SstI/NdeI) and thepar locus fragment (AatII/NdeI) are mixed and ligated to form pCFM636.

E. pCFM736

Plasmid pCFM736 is prepared as a derivative of pCFM636 and includes aforeshortened P_(L) promoter sequence. The plasmid is prepared bydigestion of pCFM636 with AatII and XbaI and insertion of the followingAatII to XbaI fragment: ##STR14## Note that this procedure deletes thesecond HpaI site present in pCFM636.

F. pCFM836

Plasmid pCFM836 is constructed as a derivative of pCFM736 for thepurpose of adding an SstII restriction site to the restriction sitebank, adding three nonsense or "stop" codons and "killing" an NcoI sitepresent in the Kanamycin resistance gene. The plasmid may be prepared bydigesting pCFM736 with BamHI and inserting the following fragment:##STR15## Destruction of the NcoI site in the Kanamycin resistance geneis accomplished by site specific mutagenesis at the codon for athreonine residue 76 amino acids upstream of the carboxy terminalleucine specified by the Kanamycin resistance gene, and specifically byaltering the ACC codon to an ACT codon.

As was the case with the Example 6 expression systems (wherein theexemplary exogenous gene inserted into the plasmids was accompanied byP_(L) promoter), expression systems involving those of the aboveplasmids which are designed to include the P_(L) promoter or shortenedP_(L) promoter will optimally require association of the C_(I857) genein the system.

Numerous modifications and variations in the invention as abovedescribed are expected to occur to those skilled in the art.Consequently, only such limitations as appear in the appended claimsshould be placed upon the invention.

What is claimed is:
 1. DNA plasmid pCFM414, A.T.C.C.
 40076. 2. DNAplasmid pCFM424.
 3. DNA plasmid pCFM510.
 4. DNA plasmid pCFM511.
 5. DNAplasmid pCFM512.
 6. DNA plasmid pCFM516.
 7. DNA plasmid pCFM526.
 8. DNAplasmid pCFM536.
 9. DNA plasmid pCFM636.
 10. DNA plasmid pCFM736. 11.DNA plasmid pCFM836.